Over-expression of DAAO and catalase in Kluyveromyces marxianus through media optimization, permeabilization and GA stabilization techniques

The selected thermotolerant, lactose-utilizing yeast strain Kluyveromyces marxianus NBIMCC 8362 possesses high specific d-amino acid oxidase activity (60 U g−1), which was increased nine-fold (545 U g−1) by design of the growth medium and conditions for d-amino oxidase induction. Applying an optimized simple and rapid procedure for chemical permeabilization of K. marxianus cells with the cationic detergent cetyltrimethylammonium bromide, the enzyme activities (d-amino acid oxidase and catalase) of the cells have been further increased for up to 43- and 58-fold, respectively. However, the enzyme activities of the permeabilized cells decreased rapidly due to the leakage of the enzymes. Treating the permeabilized cells with 0.1% glutaraldehyde at 4 °C for 10 min stabilized the enzyme in the cells and prevented their outflow. The process is stable for 10 cycles and the productivity measured was 16.6 mmmol l−1 h−1. The d-alanine transformation efficiency of K. marxianus permeabilized and GA entrapted cells was 98%.